Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques

Severe COVID-19 has been associated with T cell lymphopenia1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.


Results. 21
All macaques were inoculated with the Washington isolate of SARS-CoV-2 as previously 22 described 7 and then rested for six weeks. The animals were then challenged a second time as 23 previously. Two separate experiments were carried out, each with three animals per group for a 24 total of six macaques per group. All results from individual animals are labeled with the same 25 symbol throughout: black symbols for animals in the first experiment and orange for those in the 26 second. Findings from the re-infection are highlighted in yellow throughout. 27 Lymphocyte responses in normal control animals. Most of the non-depleted control animals 28 showed a rapid but transient lymphopenia with loss of CD4 + T helper cells, CD8 + T cells and 29 also B cells from the blood, possibly due to homing to lymphoid tissues. CD4 + T numbers 30 rebounded to approximately equivalent or higher levels by 7 dpi (Fig. 1b) and CD8 + T cell 31 counts were significantly higher at 7 dpi than at day 0 ( Fig. 1c) suggesting mobilization or a 32 proliferative response to infection. In support of a proliferative response there was a significant 33 increase in Ki-67 expression at 7 dpi (Supplementary data Fig. 1a). Similar responses were 34 observed following re-infection (yellow shading). B cell numbers in the blood also decreased 35 rapidly after infection and then rebounded over the next several weeks (Fig. 1d). 36 Lymphocyte responses in CD4-depleted animals. At day 0 the CD4 + T cell depletion in the 37 blood was greater than 90% in all but one animal (#CD4-5 was 78% depleted) (Fig. 1e). No 38 significant increases in numbers were observed in the six subsequent weeks suggesting that little 39 or no immunological priming occurred. Most animals showed slight increases in CD4 + T cell 40 counts following re-infection, but it is not known if this was a response to infection or simply 41 reconstitution following depletion (Fig. 1e, yellow shading). The CD8 + T cell responses to 42 infection were quite similar to the controls (Fig. 1f)  In summary, there was no major impact of T cell depletions on the antibody responses even 149 though there was no detectable B cell response in the blood of the CD4-depleted animals (Fig.  150 1g). The binding and neutralizing antibody titers waned by 42 dpi but rapidly expanded upon re-151 challenge regardless of T cell depletions. Thus, the memory B cell response may be more 152 critical for protection from re-challenge than standing high titers of antibody. 153 Cervical lymph node analysis. T cell levels in blood may not reflect levels in tissues where 154 cells may be more refractory to antibody-mediated depletions. No biopsies were taken during the 155 course of the experiment, but immunohistochemical (IHC) staining was used to examine CD4 + 156 and CD8 + T cells in necropsy tissues at the termination of the experiment (56 dpi). A 157 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; https://doi.org/10.1101/2021.04.02.438262 doi: bioRxiv preprint representative section from a cervical lymph node (LN), which drains the upper respiratory tract 158 is shown for each experimental group (Fig. 5). 159 CD4 + T cells. Sections of cervical lymph nodes (LN) from controls showed abundant CD4 + T 160 cells in periarteriolar lymphoid sheaths and the mantle zone of lymphoid follicles. Representative 161 low and high-magnification sections are shown in Fig. 5a. In comparison, CD4 + T cells were 162 greatly reduced in the lymphoid follicles from CD4-depleted animals, although not completely 163 gone (Fig. 5b). CD4 + T cell staining appeared normal in the CD8-depleted animals (Fig. 5c), but 164 the CD4/8-depleted animals showed moderately decreased staining (Fig. 5d). 165 Immunohistochemical staining of spleen sections gave results very similar to the cervical lymph 166 nodes (Supplementary data Fig. 5a, b). These findings cannot distinguish between T cells never 167 depleted and re-seeding of the tissues during the course of the experiment. 168 CD8 + T cells. Compared to the control (Fig. 5e), the CD8 + T cell staining in the CD4-depleted 169 animals appeared normal to slightly increased (Fig. 5f). The LNs from both the CD8-depleted 170 and dually depleted animals showed dramatically reduced CD8 + T cell staining (Fig. 5g, h). 171 B cells. The LN were also stained with anti-CD20 to detect B cells. Unlike the diminished levels 172 of B cells in the blood of CD4-depleted animals (Fig. 1g), no difference in total CD20 staining 173 was observed in the LN (Fig. 5i-l). 174 175

Discussion. 176
It has been previously reported that rhesus macaques infected with SARS-CoV-2 are 177 resistant to severe disease, similar to most young adult humans 7 . In this study we investigated 178 the role of CD8 + T cells in the natural resistance of macaques to severe COVID-19. The 179 relevance of the results is highlighted by the fact that T cell lymphopenia and T cell dysfunction 180 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; https://doi.org/10.1101/2021.04.02.438262 doi: bioRxiv preprint are associated with severe illness in humans with SARS-CoV-2 infection 1,2, 12 5,13 . Interestingly, 181 we found that depletion of either CD4 + and CD8 + T cells only slightly prolonged the recovery of 182 macaques from a first infection with SARS-CoV-2 and had little or no impact on recovery from 183 re-infection. This is not to say that T cells do not normally play roles in controlling acute SARS-184 CoV-2 infections or anamnestic responses, but rather that such roles do not appear critical or that 185 their loss may be compensated by other immune cells in macaques. On the other hand, there was 186 also no evidence that T cells played a pathological role in the development of disease in 187 macaques, as the disease severity in the T cell replete animals was similar to the T cell-depleted 188

animals. 189
It was unexpected that the CD4-depleted animals would class switch to Ig during primary 190 infection and also mount anamnestic IgG responses upon second infection. The SARS-CoV-2 191 virion does not contain classic T cell-independent antigens, which are typically rigid arrays of 30 192 or more repeated epitopes optimally separated by 5-10 nm such as those present on vesicular 193 stomatitis virus 14 15 . However, there is evidence that SARS-CoV-2 patients mount 194 extrafollicular B cell responses 16,17 , a type of response that has been shown in mouse studies to 195 produce IgM memory B cells and also IgG class-switched memory B cells in a T cell-196 independent manner 18, 19 . In that regard most of the mice in all of the groups in this study 197 mounted anamnestic IgM responses (Fig. 4 a, d, g, j), and most also produced inflammatory 198 CXCL-10 ( Supplementary Fig. 6), a biomarker associated with severe COVID-19 in humans, 199 and also associated with extrafollicular B cells in COVID-19 patients 17 . That said, our analysis 200 did not definitively demonstrate the presence of extrafollicular B cells. We cannot exclude the 201 possibility that despite high level depletions in the blood (Fig. 1d), sufficient CD4 + T cells 202 remained present in lymphoid tissues to generate immunological help. However, the diminished 203 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; https://doi.org/10.1101/2021.04.02.438262 doi: bioRxiv preprint CD4 staining in cervical lymph nodes at 56 dpi (Fig. 5B) and the strongly ablated B cell 204 responses observed in the blood of the CD4-depleted animals compared to controls (Fig. 1g) 205 argue that the CD4 depletions had a major impact on the T cell repertoire of the animals. B cell 206 activation and stimulation of proliferation typically occur in secondary lymphoid organs where 207 they interact with helper T cells 20 , but the B cells then disperse and appear in the blood at least 208 transiently, as observed in the control animals but not the CD4-depleted animals (Fig. 1d). It is 209 also possible that help for immunoglobulin class switching was provided by compensatory 210 responses from non-CD4 + cells. For example, in the absence of CD4+ T cells, mouse studies 211 with inactivated influenza virus revealed that CD4 − CD8 − double-negative αβ T cells could 212 provide help for immunoglobulin class switching 21 . In addition, interferon gamma-producing γδ 213 T cells have also been shown to enable compensatory immunoglobulin class switching 22 . 214 In McMahan et al. 11 rhesus macaques previously infected with SARS-CoV-2 were 215 depleted of CD8 + T cells prior to re-infection, which occurred at 7 weeks after the first infection. 216 In that case all 5 of the depleted animals showed breakthrough virus in nasal swabs whereas only 217 one of the controls did. This finding led to the conclusion that CD8 + T cell depletion abrogated 218 the recovery of convalescent macaques. Our study found a slightly prolonged recovery from the 219 first infection but not from re-infection due to CD8 + T cell depletion. In our experiments, which 220 employed the same Washington virus isolate and depleting antibody, half of the control animals 221 and half of the CD8-depleted animals showed breakthrough virus in nasal swabs following re-222 infection as measured by sgRNA (Fig. 2). Experimental differences included the timing of the re-223 challenge and the CD8 depletions, as well as the addition of the ocular route of challenge in our 224 experiments. While we found less effect on reinfection than the McMahan study, the difference 225 between these two small studies is not statistically different. Both our studies indicate a role for 226 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; https://doi.org/10.1101/2021.04.02.438262 doi: bioRxiv preprint

CD8 + T cells but a more important role for virus-specific antibodies. It would be of interest in 227
further studies to determine whether T cell immunity in the absence of antibody responses would 228 be sufficient for protection. 229 Old age in humans plays a significant role in susceptibility to severe disease and death 230 from SARS-CoV-2 infections, and it is well known that advanced age is associated with 231 immunosenescence 23 and dysregulated inflammatory responses 24 that result in increased 232 vulnerability to infectious diseases. Thus, aged individuals are likely to be considerably less able 233 than adult macaques to compensate for immune deficiencies such as SARS-CoV-2-induced 234 lymphopenia. The oldest macaque in our study was 9 years old, which is not considered to be 235 aged. It has been shown that aged macaques develop lower antibody responses to SARS-CoV-2 236 infections 25,26 , so is possible that different results would have been obtained using aged 237 macaques. That said, our results from adult macaques with profound depletion of either CD4 + or 238 CD8 + T cells leads to the conclusion that neither subset played a critical role in their recovery 239 from acute disease or re-infection. On the other hand, anamnestic antibody responses were 240 strongly associated with recovery from a second infection and reduced disease. 241 The current studies do not address the requirement for T cells in long-term memory and 242 protection from COVID-19, which will require further experiments. We are currently faced with 243 the emergence of multiple SARS-CoV-2 variants containing mutations in RBD that allow partial 244 or total escape from monoclonal antibodies and reduce vaccine-induced virus neutralization by 245 antibodies 27,28 . Furthermore, vaccines based on the original spike sequence appear less 246 protective against the new variants, particularly the South African B.1.351 variant. It is not 247 known whether emerging variants are also evolving to escape T cell responses, but the current 248 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021.  CoV-2 by four routes as previously described 7 : intratracheal (4 ml), intranasal (0.5 ml each 315 nostril), oral (1 ml) and ocular (0.25 ml each eye). Back titrations for the inocula showed titers of 316 3.16x10 5 50% tissue culture infectious dose (TCID50)/ml for the first challenge of the first 317 experiment, 4.3x10 5 TCID50/ml for the second challenge, 4.4x10 5 TCID50/ml for the first 318 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; https://doi.org/10.1101/2021.04.02.438262 doi: bioRxiv preprint challenge of the second experiment and 4.3x10 5 TCID50/ml for the second challenge of the 319 second experiment. SARS-CoV-2 isolate nCoV-WA1-2020 (MN985325.1)14 (Vero passage 3) 320 was provided by the Centers for Disease Control and Prevention, and propagated as previously 321 described 7 . 322 Clinical Exams and necropsy. Macaques were monitored for clinical signs at least twice daily 323 throughout the experiment using a standardized scoring sheet as previously described 7 . On exam 324 days (Fig. 1A), clinical parameters such as bodyweight, body temperature and respiration rate 325 were collected, as well as ventrodorsal and lateral chest radiographs. Chest radiographs were 326 interpreted in a blinded manner by a board-certified clinical veterinarian. The total white blood 327 cell count, lymphocyte, neutrophil, platelet, reticulocyte and red blood cell counts, and 328 hemoglobin and hematocrit values were determined from EDTA-treated blood using an IDEXX 329 ProCyte DX Analyzer (IDEXX Laboratories). Necropsies were performed after euthanasia and 330 gross pathology was scored by a board-certified veterinary pathologist. Histopathological 331 analysis of tissue slides was performed by a board-certified veterinary pathologist blinded to the 332 group assignment of the macaques. 333 Histology and Immunohistochemistry. Tissues were fixed in 10 % Neutral Buffered Formalin 334 with two changes, for a minimum of 7 days according to IBC-approved SOP. Tissues were 335 processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded 336 series of ethanol, xylene, and PureAffin. Embedded tissues were sectioned at 5 μm and dried 337 overnight at 42°C prior to staining with hematoxylin and eosin. Specific staining was detected 338 using SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 339 1:1000 dilution, CD4 antibody (abcam cat#ab133616) at a 1:100 dilution, CD8 antibody (Sino 340 Biological cat#10980-T24) at a 1:500 dilution, and CD20 (Thermo Scientific cat#RB-9013) at a 341 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. pathology. Days 0 and 42 radiographs were taken prior to inoculation, and thus serve as a 357 baseline for each animal. As such, scores for all lung lobes on Day 0 were set to "0 = normal 358 examination." All subsequent radiographs were compared to the Day 0 radiographs, evaluated 359 for changes from baseline and scored based on the criteria noted above. At study completion, 360 thoracic radiograph findings are reported as a single radiograph score for each animal on each 361 exam day. To obtain this score, the scores assigned to each of the six lung lobes are added 362 together and recorded as the radiograph score for each animal on each exam day. Scores 363 therefore range from 0 to 18 for each animal on each exam day. 364 and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. 175 flasks using the calcium phosphate method, then were concentrated by ultracentrifugation in 382 a sucrose cushion 31 . We utilized A549-ACE2 cells 32

as target cells as these cells attached better 383
to the culture plate compared to 293T-ACE2 cells 30 , allowing for extensive washes given that 384 the nanoluciferase reporter yielded high backgrounds. A549-ACE2 cells were cultured in 385 complete media containing F-12 Ham's Media (Corning), 10% fetal bovine serum (Atlanta 386 Biologicals) and 1% penicillin/streptomycin/glutamine (Corning). For the Nab assay, a 387 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. were co-incubated at 37°C for 1 h with a nonsaturating dose of the SARS-CoV-2 USA-404 WA1/2020 strain that would yield ~100,000 copies in the quantitative PCR assay. After 24 h, 405 virus copy numbers were evaluated from culture supernatant using nucleocapsid-specific primers 406 and probes as we previously described 32 . 407 Virus detection. RNA was extracted from swabs and bronchoalveolar lavage using the QiaAmp 408 Viral RNA kit (Qiagen) according to the manufacturer's instructions and as described 7 . 5 μl 409 RNA was used in a one-step real-time RT-PCR E assay using the Rotor-Gene probe kit (Qiagen) 410 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; T cell subset-depleting antibodies were administered on days -7, 0 and +4 as indicated by the blue arrows. Infections were done on days 0 and 42 as indicated by red arrows. Blood withdrawals were performed on the days indicated by the black arrows and flow cytometry was used to determine the lymphocyte subset numbers over time. The flow cytometry gating strategies are shown in Supplemental data Fig.  1b. Each symbol represents a single animal throughout. All CD4-depleted animals except CD4-5 were still greater than 90% depleted of CD4 + T cells at 7dpi. CD4-5 was 78% depleted. CD4+ Th numbers excluded FoxP3 + cells. At 7 days post-re-infection (49 dpi) the animals averaged 81% depleted. All CD8-depleted animals were >99% depleted at 7dpi and remained 78% depleted at 49dpi. The differences between subset numbers at 0 dpi and 7 dpi were calculated by a two-way paired t test. ns = not significant and other p values are shown. Numbers of B cells (d, g, j, m) were determined by flow cytometry using CD45 and CD20 as markers. The numbers of B cells in the CD4-depleted group were significantly lower over time than the controls as determined by mixed effects analysis (p=0.0118).

Figure 2. Virus detection from nasal swabs and Broncho-alveolar lavages. (a-d) Each
symbol represents the value of viral RNA copies from an individual animal at each time point. The brackets delineate comparisons of cumulative values between the first two weeks after infection with the two weeks after re-infection, and numbers indicate the p values from two-way paired t tests showing significantly reduced virus levels following the second infection (b, c, d) except in the control group (a), which was marginally non-significant. The cumulative RNA titers from the CD4-and CD8-depleted groups for the first two weeks after initial infection were not significantly different than the controls but the CD4/CD8-depleted group had significantly higher titers (p = 0.0362 by one-way Anova with a Dunnett's post-test). b In (e-h) mean values are shown comparing the total viral RNA data from a -d (black lines) with sub-genomic sgRNA results (blue lines). (i-l) Broncho-alveolar lavage fluids were taken at one day after infection and re-infection. sgRNA was measured from each animal and showed significantly reduced virus replication upon second infection (p values from paired t tests shown in figure). and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; https://doi.org/10.1101/2021.04.02.438262 doi: bioRxiv preprint Figure 3. Clinical and radiograph scores and neutrophil counts. (a -d) Clinical signs were scored in a blinded manner using a clinical score sheet. Cumulative scores for the two weeks following the first infection were compared with the two weeks following re-infection (yellow shading) using a two-way paired Student's t test. The dashed lines indicate means. (e -h) Radiographs were scored in a blinded manner for the presence of pulmonary infiltrates by two board-certified clinical veterinarians. Cumulative scores were analyzed as in a-d. The dotted line indicates the maximum score if all lobes were severely affected. (il) Neutrophil counts for each animal were taken on blood withdrawal days and significant differences were noted only between days 0 to 3 as shown. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Figure 4. Antibody responses. SARS-CoV-2 spike receptor binding domain-specific IgM (a, d, g, j) and IgG responses (b, e, h, k) were assayed in individual macaques over the course of the experiment using the Mesoscale kit as described in the methods. Each symbol represents a specific animal throughout. Neutralizing antibody titers (c, f, I, l) were measured using a lentiviral pseudovirus expressing the human SARS-CoV-2 spike as described in the method section. Values are reciprocal dilutions that produced an 80% reduction in pseudovirus infection (c, f, i, l). Statistics comparing titers at days 7 dpi and 49 dpi were done using a two-way paired Student's t test * = p < 0.05, **** = p < 0.0001. Unlabeled comparisons were not statistically significant. Analysis of differences between experimental groups was done by one-way Anova with a Dunnett's multiple comparisons post-test. The ability of sera from 28dpi to neutralize live SARS-CoV-2 confirmed results from the pseudovirus neutralization assay (Supplemental data Fig. 4). and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 4, 2021. ; SARS-CoV-2 N1 (copies/5ul input)

SARS-CoV-2 neutralization
and is also made available for use under a CC0 license.